The Respiratory‐Chain NADPH Dehydrogenase ofAzotobacter vinelandii

Abstract

I. NADPH dehydrogenase activity of Azotobacter vinelandii respiratory membranes is inhibited by adenine nucleotides (pure competitive inhibition; AMP>ADP>ATP) and by NAD+ (partially competitive, allosteric inhibition). No allosteric activators, other than NADPH, were found for this enzyme.2. The dehydrogenase exhibits kinetic properties compatible with the presence of a catalytic site, which binds one molecule of NADPH (or one molecule of adenine nucleotide), and an apparently acid-sensitive, site (or sites) which binds one molecule of either NADPH (positive homotropic effector) or NAD+ (negative heterotropic effector).3. These results suggest that in the presence of NAD+ or low energy charge, NADPH may be converted to NADH via the NADPH-tNAD+ transhydrogenase, and thence oxidised via the more efficiently phosphorylating NADH dehydrogenase.4. The prime function of the NADPH dehydrogenase is probably to oxidise tJhe excessNADPH which might be expected to accumulate when nitrogen-fixation is switched off (e.g. under highly aerobic conditions) and thus to contribute to the phenomenon of nitrogenase protection by enhanced respiration and also to the maintenance of the desired intracellular NADPH/NADP+ ratio.The membrane-bound NADH, L-malate and NADPH dehydrogenases are the rate-limiting, initial enzymes in the oxidation of their respective substrates by the highly active respiratory chain of Azotobacter vinelandii [1,2]. It might be expected therefore that any control over respiration would be exerted through these rate-limiting dehydrogenases. This is indeed the case with malate dehydrogenase, the activity of which in vitro is determined by the concentration of its product oxaloacetate (unpublished results) and with NADH dehydrogenase where the presence of site-I phosphorylation [4,5] enables the activity of this enzyme complex to be regulated, in the presence of inorganic phosphate, by the availability of the phosphate acceptor ADP [4,6,71.

Hyperproduction of Poly-β-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

“…In the course of genetic transformation experiments, it was noted that A. vinelandii UW frequently produced rifampin-resistant (Rif) transformants of various colony sizes when transformed with A. vinelandii 113 DNA. When these colonies were left at room temperature for approximately 1 week, the variations in colony size persisted, but some of the large colony transformants had a sectored appearance. These sectored colonies had distinct white rays within the normal pinkish tan A. vinelandii cell mass.…”

Respiratory protection of nitrogenase in Azotobacter vinelandii

“…Since highly aerobic conditions lead to the "switching-off' of nitrogenase [4, 51 and hence to an increased availability of NADPH for oxidation via the non-phosphorylating NADPH dehydrogenase [8], excess oxygen clearly promotes the utilisation of respiratory pathways of relatively low energy conservation efficiency in A. vinelandii.…”

Encystment and germination in Azotobacter vinelandii.

“…This protein catalyzes the reversible transfer of hydrogen between NAD(P)H and NAD(P)+; it has a flavine adenine dinucleotide (FAD) cofactor, and, although its minimal molecular weight is 60,000, it forms aggregates whose sedimentation coefficients are 24, 48, and 88S (164). This respiratory regulating enzyme in combination with the membrane-bound NADH (127,163) and NADPH dehydrogenases (1,120) controls the NAD(P)H to NAD(P)+ ratios in the cell.…”